Reinforcement-guided generative protein language models enable de novo design of highly diverse AAV capsids
Authors: Lucas Ferraz, Ana F. Rodrigues, Pedro Giesteira Cotovio, Mafalda Ventura, Gabriela Silva, Ana Sofia Coroadinha, Miguel Machuqueiro, Catia Pesquita
Organizations: LASIGE, Faculdade de Ciências da Universidade de Lisboa, Lisboa, Portugal · iBET – Instituto de Biologia Experimental e Tecnológica, Oeiras, Portugal · BioISI, Faculdade de Ciências da Universidade de Lisboa, Lisboa, Portugal
Adeno-associated viral (AAV) vectors are widely used delivery platforms in gene therapy, and the design of improved capsids is key to expanding their therapeutic potential. A central challenge in AAV bioengineering, as in protein design more broadly, is the vast sequence design space relative to the scale of feasible experimental screening. Machine-guided generative approaches provide a powerful means of navigating this landscape and proposing novel protein sequences that satisfy functional constraints. Here, we develop a generative design framework based on protein language models and reinforcement learning to generate highly novel yet functionally plausible AAV capsids. A pretrained model was fine-tuned on experimentally validated capsid sequences to learn patterns associated with viability. Reinforcement learning was then used to guide sequence generation, with a reward function that jointly promoted predicted viability and sequence novelty, thereby enabling exploration beyond regions represented in the training data. Comparative analyses showed that fine-tuning alone produces sequences with high predicted viability but remains biased toward the training distribution, whereas reinforcement learining-guided generation reaches more distant regions of sequence space while maintaining high predicted viability. Finally, we propose a candidate selection strategy that integrates predicted viability, sequence novelty, and biophysical properties to prioritize variants for downstream evaluation. This work establishes a framework for the generative exploration of protein sequence space and advances the application of generative protein language models to AAV bioengineering.
Adeno-associated viruses (AAVs) are promising vectors for gene therapy, but their native serotypes face limitations in tissue tropism, immune evasion, and production efficiency. Engineering capsids to overcome these hurdles is challenging due to the vast sequence space and the difficulty of simultaneously optimizing multiple functional properties. The complexity also adds when it comes to the kidney, which presents unique anatomical barriers and cellular targets that require precise and efficient vector engineering. Here, we present AAVGen, a generative artificial intelligence framework for de novo design of AAV capsids with enhanced multi-trait profiles. AAVGen integrates a protein language model (PLM) with supervised fine-tuning (SFT) and a reinforcement learning technique termed Group Sequence Policy Optimization (GSPO). The model is guided by a composite reward signal derived from three ESM-2-based regression predictors, each trained to predict a key property: production fitness, kidney tropism, and thermostability. Our results demonstrate that AAVGen produces a diverse library of novel VP1 protein sequences. In silico validations revealed that the majority of the generated variants have superior performance across all three employed indices, indicating successful multi-objective optimization. Furthermore, structural analysis via AlphaFold3 confirms that the generated sequences preserve the canonical capsid folding despite sequence diversification. AAVGen establishes a foundation for data-driven viral vector engineering, accelerating the development of next-generation AAV vectors with tailored functional characteristics.
Regenerative Medicine Research Center, Isfahan University of Medical Sciences, Isfahan, Iran · Department of Genetics and Molecular Biology, Isfahan University of Medical Sciences, Isfahan, Iran
Protein language models are standard priors for biological sequence generation, but steering them toward explicit distributional design targets remains largely unexplored. We study a constrained protein generation problem in which sequences must match a desired amino-acid (AA) composition profile while preserving plausible sequence statistics and diversity. The motivating application is synthetic feed protein design, where the AA composition of dietary proteins directly determines their nutritional value. We propose a two-stage pipeline in which domain-adaptive fine-tuning (FT) on an in-domain protein dataset is followed by iterative reward-weighted FT via reinforcement learning (RL) anchored against the FT model as a frozen reference. We evaluate the pipeline on two AA compositions and find that FT brings the average composition close to the target, while the subsequent RL enforces specific sequence constraints that FT alone cannot satisfy. We additionally evaluate the design choices of the proposed composition reward term against two baselines and an ablated variant, isolate the contribution of each training stage, and verify that AA composition alignment is achieved without degrading sequence quality.
Violeta Basten-Romero, Rubén Muñoz-Tafalla, Anna María Díaz-Rovira +3
Barcelona Supercomputing Center (BSC), Plaça d’Eusebi Güell, 1-3, 08034, Barcelona, Spain · Department of Bioinformatics, School of Mathematics and Statistics (FME), Universitat Politècnica de Catalunya - BarcelonaTech (UPC), 08034 Barcelona, Spain · Nostrum Biodiscovery S.L., Av. Josep Tarradellas, 8-10, 3-2, 08029, Barcelona, Spain +1
Generative modeling has become a central paradigm in protein research, extending machine learning beyond structure prediction toward sequence design, backbone generation, inverse folding, and biomolecular interaction modeling. However, the literature remains fragmented across representations, model classes, and task formulations, making it difficult to compare methods or identify appropriate evaluation standards. This survey provides a systematic synthesis of generative AI in protein research, organized around (i) foundational representations spanning sequence, geometric, and multimodal encodings; (ii) generative architectures including SE(3)-equivariant diffusion, flow matching, and hybrid predictor-generator systems; and (iii) task settings from structure prediction and de novo design to protein-ligand and protein-protein interactions. Beyond cataloging methods, we compare assumptions, conditioning mechanisms, and controllability, and we synthesize evaluation best practices that emphasize leakage-aware splits, physical validity checks, and function-oriented benchmarks. We conclude with critical open challenges: modeling conformational dynamics and intrinsically disordered regions, scaling to large assemblies while maintaining efficiency, and developing robust safety frameworks for dual-use biosecurity risks. By unifying architectural advances with practical evaluation standards and responsible development considerations, this survey aims to accelerate the transition from predictive modeling to reliable, function-driven protein engineering.
Senura Hansaja Wanasekara, Minh-Duong Nguyen, Xiaochen Liu +2
Biomedical Engineering, The University of Sydney, Darlington, NSW 2006, Australia. · the College of Engineering and Computer Science, VinUniversity, Vietnam. · School of Computer Science and School, The University of Sydney, Darlington, NSW 2006, Australia.